The invention relates to a rapid method of detecting pathogen or prion protein that may be use to determine the clearance of pathogen protein, in general, and to a Western blot immunoassay method of relating pathogen protein clearance to infectivity clearance, in specific. The method has been applied to the quantitation of TSE protein clearance and its relationship to infectivity clearance.
The Cohn-Oncley purification of therapeutic proteins from blood plasma, referred to herein in general as the Cohn process or scheme, employs a series ethanol additions and pH adjustments to purify or enrich for proteins which may be used in human therapies. Commonly purified proteins include immunoglobulins, anti-hemophiliac factors and albumin. While many manufacturers of such products utilize the basic Cohn scheme, frequently established steps may be modified or additional steps are implemented to increase either the purity and/or yield for a given product. Such steps are typically proprietary for a given manufacturer.
Since the discovery that HIV could be carried and transmitted though the use of blood products, the interest and concern about the presence of such pathogenic agents in biological products derived from blood has increased. Most recently, there has been concern that CJD, Creutzfeldt-Jakob Disease, could be transmitted through the use of blood-derived product. CJD is one of the human transmissible spongiform encephalopathies (TSE), a collection of neurodegenerative diseases that are debilitating and fatal. Infectivity associated with CJD appears to be either associated with or caused by the prion protein (PrP). Although new disease carrying viruses may be generated at any time, manufacturers of blood-based products take precautions to obtain a blood product that is free of known transmissible diseases, to the extent for which these can be tested. Unfortunately, the primary test for possible TSE infectivity is a biological assay in which rodents are injected with the material of interest to see if infectivity develops. The results of such assays require nine months to a year to develop, frequently too long to hold a manufactured lot of plasma product prior to release for use.
Therefore, a method of detecting a protein associated with a pathogen suspected of carrying infectivity such as the prion or viral surface (coat) protein is important for the blood fractionation industry. A rapid, sensitive method capable of determining the removal of virus or pathogenic prion protein would provide the blood fractionation industry with a useful tool for determining what danger of infectivity exists after a particular manufacturing process step. The decrease in viral or prion protein relative to a given product associated with a manufacturing process step is referred to herein as xe2x80x9cclearancexe2x80x9d. Because of the importance of such a test for TSE infectivity to the safety of plasma products, the method of this invention was described generally at the Blood Safety and Screening conference held in McClean, Va. on Feb. 23, 1998.
The invention is an immunoassay method of detecting viruses or prion protein content of a biological sample. This method provides a quantitative measure of the viral or prion protein content that may be related to infectivity. The method an typically detect a range of prion protein from 3 logs to 5 logs dynamic range and the measured clearance correlates well with infectivity clearance for the process steps have been tested. The preferred immunoassay method is a Western blot and results are available in 2-4 days. The method is particularly useful to track the prion protein related to potential infectivity in plasma production.
The method of the invention is composed of the steps of preparing: a) a biological sample, usually a plasma or plasma manufacturing intermediate sample, for an immunoassay, either a Western blot immunoassay or an ELISA immunoassay; b) performing the immunoassay for the protein associated with infectivity; c) quantitating the protein results; and, d) relating the protein results to infectivity. Preferably the quantitative method employs a Western blot immunoassay method. This method may be used to determine clearance of the pathogenic protein in a biological sample such as a plasma product or plasma processing sample by preparing an aliquot of a first sample; performing the Western blot assay on such first sample; quantitating the pathogenic protein results in the first sample; processing the first sample to obtain a second sample or samples from the process stream of the first sample; performing and quantitating the pathogenic protein results from the second sample or samples and comparing relative amounts of pathogenic protein detected in the first and second sample to determine the clearance of the processing step.
An application of particular interest is determining the clearance of TSE by a particular plasma processing step. The comparative quantitative results of the two immunoassays provide a measure of the xe2x80x9cclearancexe2x80x9d obtained by the processing step.
The preferred method is composed of the following steps:
a. spiking a process solution with a brain homogenate from an animal infected with the pathogen marker (typically a protein) of interest;
b. processing the spiked solution; and
c. assaying for the presence of the pathogen protein marker (prion protein) in the resulting fractions for distribution of the protein.
The assay step is composed of the following steps:
a. taking a sample of each fraction of interest;
b. diluting the samples in defined increments;
c. treating each diluted sample with proteinase-K;
d. (optionally) centrifuging the proteinase-K treated samples; and
e. performing a Western blot or ELISA immunoassay.
The preferred Western blot immunoassay is composed of the following steps:
a. separating the proteinase K treated samples electrophoretically;
b. transferring the separated samples to a membrane;
c. adding a blocking agent to the membrane containing the separated samples;
d. incubating the membranes with a first antibody capable of binding the pathogenic protein;
e. washing the incubated membrane with a low salt buffer to remove any non-binding antibodies and proteins;
f. incubating the washed membrane with a second antibody capable of recognizing the first antibody, which second antibody contains a reporter group capable of providing a measurable signal; and
g. measuring the signal produced by counting the number of lanes with detectable signal.
The number of lanes with detectable protein from sample diluted in defined increments allows for the estimation of infectivity clearance for a sample when compared to the spiked input material (prove). For the TSE protein assay, a preferred first antibody is the monoclonal antibody, 3F4.